Easy quantification of template-directed CRISPR/Cas9 editing

Abstract

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations ingenomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/218156 doi: bioRxiv preprint first posted online Nov. 13, 2017; 2 The CRISPR system for genome editing has become one of the most popular techniques in molecular biology. CRISPR endonucleases such as Cas9 can cleave genomic DNA with high precision, and due to error-prone repair mechanisms this can result in small insertions or deletions (indels) [1][2][3] .Alternatively, precisely designed small nucleotide changes can be incorporated near the break site by providing a donor template 4,5 , such as a single-stranded oligodeoxynucleotide (ssODN) 4,6 . By homology-directed repair (HDR), the DNA of the donor template is exchanged with the genomic DNA, and thereby the desired mutations are introduced 7,8 . Such precise editing offers the possibility to create and study specific mutations, or to correct disease-causing nucleotide variants 5,9 .A current limitation of this template-directed strategy is that the efficacy is unpredictable and often low. Because error-prone non-templated repair pathways are active besides HDR, various indels are often introduced at the target site instead of the desired mutation. Moreover, a substantial fraction of the target sequence may remain unaltered. Thus, exposing a pool of cells to CRISPR and a donor template yields a complex mixture of cells with wild-type DNA, indels and the designed mutation, with unpredictable ratios [10][11][12] . A quick and easy assay to determine these ratios is of key importance, particularly if one wants to estimate how many cells are to be cloned from the pool in order to obtain at least one clonal line with the desired mutation.High throughput sequencing of DNA around the induced break site is a powerful tool to analyze the mutation spectrum 13 , but is also expensive and requires substantial computational analysis. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/218156 doi: bioRxiv preprint first posted online Nov. 13, 2017; 3 the sgRNA is used as input to determine the expected break site. Indels are then quantified by computational decomposition of the mixture of sequences in the test sequence trace, using the control sequence trace for comparison 14 .TIDER takes a similar decomposition approach, but it requires one additional capillary sequencing trace. This "reference" trace is derived from a pure DNA sample that c...

Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER

“…Note that TIDE can only detect overall indel frequencies, but not nucleotide substitutions or specifically designed indels. For the latter purpose we developed TIDER (Tracking of Insertions, DEletions, and Recombination events) [14]. This method can estimate the incorporation frequency of template-directed mutations, including point mutations, and distinguish them from a background of additional indels that originate from competing erroneous repair pathways.…”

“…In general, TIDER is able to discriminate "naturally" occurring deletions and insertions from templated "designed" indels. Only in the presence of a small designed deletion (À1, À2) near the expected break site the designed mutation may be underestimated [14]. In case the designed mutation consists of an insertion larger than þ1, TIDER does not consider natural insertions of the same size, because the decomposition becomes less robust.…”

Diminished Adherence of Biomphalaria Glabrata Embryonic Cell Line to Sporocysts of Schistosoma Mansoni Following Programmed Knockout of the Allograft Inflammatory Factor

“…Amplicons were isolated using the PCR cleanup and gel extraction kit (ClonTech, Takara USA, Mountain View, CA) and the nucleotide sequence of amplicons determined by Sanger direct sequencing (GENEWIZ, South Plain eld, NJ). Chromatograms of the sequence reads from the control and experimental groups in each replicate experiment were subjected to online analysis using the TIDE algorithm, https://tide.deskgen.com/ [56,57] and also using the Inference of CRISPR v2 Edits analysis (ICE) software, https://ice.synthego.com/#/ (Synthego Corporation, Redwood City, CA) [58]. Estimates of CRISPR e ciency, insertion-deletion (INDEL)-substitution percentages, and the nucleotide sequence of mutant alleles were obtained using both the TIDE and the ICE platforms ( Fig.…”

“…The nucleotide sequence of the amplicons was determined by Sanger direct sequencing using the same primers, AIF-F and AIF-R. Both forward and reverse DNA sequencing reads were analyzed for programmed CRISPR/Cas9-catalyzed mutations using two CRISPR editing software packages, the ICE and the TIDE tools [56,57]. Both parse the Sanger sequencing les, either one at a time or as a batch of several technical replicates from the same sample, and identify the sequences complimentary to the gRNA.…”

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

“…sequencing or next-generation sequencing (NGS) enable the detection of precise genomic changes without the use of genomic markers (Brinkman et al, 2014;Pinello et al, 2016). However, Sanger-sequencing-based approaches suffer from low sensitivity and precision because of the variable quality of the sequencing reactions and background signals that often affect the sequencing reads (Brinkman et al, 2014(Brinkman et al, , 2018. Furthermore, NGS-dependent detection strategies, while highly sensitive (Clement et al, 2019;Lindsay et al, 2016;Pinello et al, 2016), remain expensive and time consuming, which limits their value for the development of mutant cell lines and animal models and for applications that require a rapid turnaround time, such as the identification of pathogenic variants in certain clinical settings.…”